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Biomedical subjects

R F Irvine

Publications and source records attributed to R F Irvine.

At least 37 records · Page 2Linked to original sources
PubMed1998

Modulation of Ins(2,4,5)P3-stimulated Ca2+ mobilization by ins(1,3,4, 5)P4: enhancement by activated G-proteins, and evidence for the involvement of a GAP1 protein, a putative Ins(1,3,4,5)P4 receptor.

We have previously shown that addition of Ins(1,3,4,5)P4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P3, and we suggested that, in doing this, Ins(1,3,4,5)P4 is not working via an InsP3 receptor but indirectly via an InsP4 receptor [Loomis-Husselbee, Cullen, Dreikhausen, Irvine and Dawson (1996) Biochem. J. 314, 811-816]. Here we have investigated whether this effect might be mediated by GAP1(IP4BP), recently identified as a putative receptor for Ins(1,3, 4,5)P4. GAP1(IP4BP) is a protein that interacts with one or more monomeric G-proteins, so we sought evidence for involvement of monomeric G-proteins in the effects of Ins(1,3,4,5)P4 in permeabilized L1210 cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) enhanced the effect of Ins(1,3,4,5)P4 on Ins(2,4, 5)P3-stimulated Ca2+ mobilization, but had no effect on the action of Ins(2,4,5)P3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P4 was also seen with GTP[S]-loaded R-Ras or Rap1a (two G-proteins known to interact with GAP1(IP4BP)), whereas H-Ras was inactive at similar concentrations. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) did not alter the action of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. Finally, the addition of exogenous GAP1(IP4BP), purified from platelets, markedly enhanced the effect of Ins(1,3,4,5)P4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P3 alone was unaltered. We conclude that the increase in Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4, 5)P4 may be mediated by GAP1(IP4BP) or a closely related protein (such as GAP1(m)), and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to cause its effect.

Animals↗
PubMed1998

Regulation of PtdIns4P 5-kinase C by thrombin-stimulated changes in its phosphorylation state in human platelets.

PtdIns(4,5)P2 production by the enzyme PtdIns4P 5-kinase C (PIPkin C) was examined in thrombin-stimulated human platelets. Thrombin caused a rapid, transient 2-3-fold increase in PIPkin activity and a transient net dephosphorylation of the enzyme. PIPkin C was phosphorylated on serine and threonine residues in unstimulated platelets; no evidence for tyrosine phosphorylation was found. The phosphatase inhibitor okadaic acid promoted PIPkin C hyperphosphorylation and a concomitant marked inhibition of its activity in immunoprecipitates. Activity was restored by treatment with alkaline phosphatase, suggesting the existence of an inhibitory phosphorylation site. In support of this idea, alkaline phosphatase treatment of PIPkin C immunoprecipitated from unstimulated platelets caused a modest (1.6-fold) but significant activation of the enzyme. However, alkaline phosphatase treatment of PIPkin C immunoprecipitated from thrombin-stimulated platelets caused a decrease in activity to approximately the same levels, suggesting that the phosphorylation of PIPkin C also contributes to the observed stimulation. Two-dimensional phosphopeptide mapping of immunoprecipitated PIPkin C revealed that the enzyme is multiply phosphorylated and that, whereas some phosphopeptides are indeed lost on stimulation, consistent with the net dephosphorylation of the enzyme, at least two novel sites become phosphorylated. This suggests that thrombin causes complex changes in the phosphorylation state of PIPkin C, one consequence of which is its activation.

Alkaline Phosphatase↗
PubMed1998

Inositide signalling in Chlamydomonas: characterization of a phosphatidylinositol 3-kinase gene.

Phosphoinositide (PI) 3-kinases, which phosphorylate the D-3 position of the inositol ring, function in several different signalling pathways. The phosphatidylinositol (PtdIns)-specific PI 3-kinase of yeast (Vps34p) is part of a receptor signalling protein complex associated with the trans-Golgi membranes, whereas PI 3-kinases that phosphorylate polyphosphoinositides in animal cells form a major receptor-controlled signalling pathway in the plasma membrane. Recent studies have indicated the presence of active PLC, PLD, and PI 3-kinase-dependent signalling systems in the unicellular green alga Chlamydomonas, and PtdIns-3P in Chlamydomonas shows a particularly high rate of turnover. Here we report the cloning of the Chlamydomonas Vps34p, and some characterisation of its properties, regulation and localisation. A single-copy 12 kb gene was present. The corresponding protein of 122 kDa had full-length homology with Vps34ps from other species, but it contained a novel spacer-like insert region of 148 amino acid residues between homology region 2 (HR2) and the C-terminal catalytic core domain, and three other shorter putative inserts. Available cDNAs were used to assemble a pBluescript clone expressing a recombinant protein which had PtdIns-specific 3-kinase activity. However, an unexpected observation was that recombinant proteins containing the complete catalytic core, but lacking HR2, had no lipid kinase activity, pointing to a previously unsuspected role for this domain, possibly in substrate binding. VPS34 mRNA and protein levels, as determined by RNAse protection assays and by immunological methods respectively, were low in all cell stages that were examined. Western blotting of subcellular fractions revealed that most of Vps34p in cell lysates of cw-15 (a cell wall-deficient mutant) could be recovered in a NP-40-resistant 100000 x g pellet, suggesting that the enzyme may have a location different from that found in higher plants.

Amino Acid Sequence↗
PubMed1997

Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei.

(1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04% (w/v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [gamma-32P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36% for DAG and 24% for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P2 did not follow the same pattern as PtdIns and DAG; after removal of the nuclear membrane, 40% of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P2 was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P2 has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [gamma-32P]ATP (70%, 90% and 94% of intact nuclei respectively), suggesting a predominantly intranuclear location for the respective kinases. (5) Nuclei also showed phosphomonoesterase and phosphatidic acid hydrolase activity; dephosphorylation of pre-radiolabelled PtdIns(4)P, PtdIns(4,5)P2 and phosphatidic acid was observed when [gamma-32P]ATP was removed. However, some of the radioactivity was apparently resistant to these enzymes, suggesting the existence of multiple pools of these lipids. (6) Addition of excess non-radiolabelled ATP to nuclei pre-labelled with [gamma-32P]ATP resulted in an initial increase in the label in PtdIns(4,5)P2, implying a precursor-product relationship between the radiolabelled pools of PtdIns(4)P and PtdIns(4,5)P2. This was confirmed by analysis of the incorporation of 32P into the 4'-phosphate group of PtdIns(4)P and the individual 4'- and 5'-phosphate groups of PtdIns(4,5)P2. The data from these experiments also indicated that PtdIns(4,5)P2 can be produced from a pre-existing pool of PtdIns(4)P, as well as de novo from PtdIns. (7) Taken together our data suggest that isolated rat-liver nuclei have an intranuclear inositol lipid metabolism mechanism utilizing enzymes and substrates equivalent to those found in cytosol and plasma membrane, and that there may be some, but not complete, compartmentalization of the components of the nuclear inositol cycle.

Adenosine Triphosphate↗
PubMed1997

Rapid kinetic measurements of 45Ca2+ mobilization reveal that Ins(2,4,5)P3 is a partial agonist at hepatic InsP3 receptors.

Ins(2,4,5)P3, a metabolically stable analogue of Ins(1,4,5)P3, is widely used in analyses of Ca2+ signalling pathways, but its utility depends upon it faithfully mimicking the effects of the natural messenger, Ins(1,4,5)P3, at InsP3 receptors. To compare the kinetics of InsP3-evoked 45Ca2+ mobilization, Ins(1,4,5)P3- and Ins(2,4,5)P3-stimulated 45Ca2+ release from the intracellular stores of permeabilized rat hepatocytes was measured using rapid superfusion. Both Ins(1,4,5)P3 and Ins(2,4,5)P3 caused concentration-dependent increases in the rate of 45Ca2+ efflux, which accelerated towards a peak and then abruptly switched to a bi-exponentially decaying release rate. However, the peak rate of 45Ca2+ mobilization evoked by maximal concentrations of Ins(2,4,5)P3 was only 65+/-3% (n = 3) of that evoked by Ins(1,4,5)P3. Furthermore, Ins(2,4,5)P3 inhibited the peak rate of 45Ca2+ efflux evoked by Ins(1,4,5)P3. These results indicate that Ins(2,4,5)P3 is a partial agonist at hepatic Ins(1,4,5)P3 receptors. Additionally, responses to Ins(2,4,5)P3 were less positively cooperative [Hill coefficient (h) = 1.9+/-0.3] than were those to Ins(1,4,5)P3 (h = 3.0+/-0.2) and the kinetics of termination of 45Ca2+ mobilization were slower. The lesser efficacy of Ins(2,4,5)P3 may account for the lower cooperativity in the responses it evokes, the slower inactivation of InsP3 receptors and the characteristic patterns of Ca2+ spiking it evokes in intact cells.

Animals↗
PubMed1996

Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton.

Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (PtdIns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp60(c-src) to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.

Blood Platelets↗
PubMed1996

Identification of diacylglycerol pyrophosphate as a novel metabolic product of phosphatidic acid during G-protein activation in plants.

We provide evidence that phosphatidic acid (PtdOH) formed during signaling in plants is metabolized by a novel pathway. In much of this study, 32Pi-labeled Chlamydomonas cells were used, and signaling was activated by adding the G-protein activator mastoparan. Within seconds of activation, large amounts of [32P]PtdOH were formed, with peak production at about 4 min, when the level was 5-25-fold higher than the control. As the level of [32P]PtdOH subsequently decreased, an unknown phospholipid (PLX) increased in radiolabeling; before activation it was barely detectable. The chromatographic properties of PLX resembled those of lyso-PtdOH and CMP.PtdOH but on close inspection were found to be different. PLX was shown to be diacylglycerol pyrophosphate (DGPP), the product of a newly discovered enzyme, phosphatidate kinase, whose in vitro activity was described recently (Wissing, J. B., and Behrbohm, H. (1993) Plant Physiol. 102, 1243-1249). The identity of DGPP was established by co-chromatrography with a standard and by degradation analysis as follows: [32P]DGPP was deacylated, and the product (glycerolpyrophosphate, GroPP) was hydrolyzed by mild acid treatment or pyrophosphatase to produce GroP and Pi as the only radioactive products. Since DGPP is the pyrophosphate derivative of PtdOH and is formed as the concentration of PtdOH decreases, we assumed that PtdOH was converted in vivo to DGPP. This was confirmed by showing that during a short labeling protocol while the specific radioactivity of DGPP was increasing, the specific radioactivity of the 32Pi derived from DGPP as above was higher than that of [32P]GroP. DGPP was also formed in suspension cultures of tomato and potato cells, and its synthesis was activated by mastoparan. Moreover, it was also found in intact tissues of a number of higher plants, for example, carnation flower petals, vetch roots, leaves of fig-leaved goosefoot, and common persicaria and microspores of rape seed. Our results suggest that DGPP is a common but minor plant lipid that increases in concentration when signaling is activated. Possible functions of DGPP in phospholpase C and D signaling cascades are discussed.

Animals↗
PubMed1996

Synergistic effects of inositol 1,3,4,5-tetrakisphosphate on inositol 2,4,5-triphosphate-stimulated Ca2+ release do not involve direct interaction of inositol 1,3,4,5-tetrakisphosphate with inositol triphosphate-binding sites.

We have previously found that for permeabilized L1210 cells, low micromolar concentrations of Ins(1,3,4,5)P4 added prior to Ins(2,4,5)P3 enhance the effects of suboptimal concentrations of Ins(2,4,5)P3 in causing Ca2+ release from InsP3-sensitive Ca2+ stores [Cullen, Irvine and Dawson (1990) Biochem J. 271, 549-553]. If this was due either to some conversion of added Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by the 3-phosphatase, or to Ins(1,3,4,5)P4 acting as a weak (or partial) agonist on the InsP3 receptor it would be expected that,in the presence of thimerosal to sensitize the InsP3 receptor, the dose-response curve to Ins(1,3,4,5)P4 would be left-shifted by the same extent as that of Ins(1,4,5)P3. This was found not to be the case; the dose-response curve to Ins(1,3,4,5)P4 was not shifted at all by thimerosal. Furthermore, L-Ins(1,3,4,5)P4, which can displace radiolabelled D-Ins(1,3,4,5)P4 but not D-Ins(1,4,5)P3 from their respective high-affinity binding sites, mimicked the effects of D-Ins(1,3,4,5)P4 in enhancing the slow phase of Ins(2,4,5)P3-stimulated Ca2+ release. Ins(1,3,4,5)P4 caused an increase in magnitude of the slow phase of InsP3-stimulated Ca2+ release leaving the magnitude of the fast phase unaltered, in contrast to increasing Ins(2,4,5)P3 concentrations which increased the size of both phases. In addition, Ins(1,3,4,5)P4 decreased the rate constant for the slow phase of Ca2+ release. These findings point strongly to the conclusion that InsP4 is not working directly via the InsP3 receptor but indirectly via an InsP4 receptor.

Animals↗
PubMed1995

Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells.

Differentiation of murine erythroleukaemia cells with the chemical agent DMSO leads to a cessation of proliferation and the production of a number of erythrocyte markers such as haemoglobin. We have previously demonstrated that activation of proliferation leads to an increase in the production of nuclear diacylglycerol (DAG). Here we demonstrate that differentiation leads to a decrease in the levels of nuclear DAG and the activity of the nuclear-associated phosphoinositidase C (PIC). The change in activity appears to be due to a decrease in the mass levels of the beta 1 isoform, as demonstrated by the use of isoform-specific antibodies. Moreover, the changes correlate with the cessation of proliferation and an increase in the number of cells in G1 phase of the cell cycle, rather than with the number of cells which have differentiated. Indeed, although treatment of the cells with phorbol 12-myristate 13-acetate (PMA) inhibits the differentiation programme as assessed by haemoglobin staining, it does not inhibit the number of cells blocking in G1 of the cell cycle or the changes in nuclear DAG or PIC activity. The possible involvement of this nuclear inositide cycle during progression through the cell cycle is discussed.

Animals↗
PubMed1995

Identification of a specific Ins(1,3,4,5)P4-binding protein as a member of the GAP1 family.

Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) is produced rapidly from inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in stimulated cells. Despite extensive experimentation, no clearly defined cellular function has yet been described for this inositol phosphate. Binding sites specific for Ins(1,3,4,5)P4 have been identified in several tissues, and we have purified one such protein to homogeneity. Its high affinity for Ins(1,3,4,5)P4, and its exquisite specificity for this isomeric configuration, suggest it may be an Ins(1,3,4,5)P4 receptor. Here we report the cloning and characterization of this protein as a GTPase-activating protein, specifically a member of the GAP1 family. In vitro it shows GAP activity against both Rap and Ras, but only the Ras GAP activity is inhibited by phospholipids and is specifically stimulated by Ins(1,3,4,5)P4.

Amino Acid Sequence↗
PubMed1995

The cloning and sequence of the C isoform of PtdIns4P 5-kinase.

In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.

Amino Acid Sequence↗
PubMed1995

Specificity of the purified inositol (1,3,4,5) tetrakisphosphate-binding protein from porcine platelets.

The specificity of the inositol 1,3,4,5-tetrakisphosphate binding protein purified from porcine platelets [Cullen et al. (1995) Biochem. J. 305, 139-143] was examined using all the isomers of myo-inositol tetrakisphosphate. From the relative potencies of these compounds it appears that phosphorylation of the 1, 3 and 5 positions is essential for high affinity binding, that there is some tolerance of phosphorylation of the 6-hydroxyl, but none of a phosphate in the 2-position, and that phosphorylation of the 4-hydroxyl has very little influence. The binding of Ins(1,3,4,5)P4 was not appreciably altered by physiological Mg2+ concentrations, and the pH dependence of binding under physiological conditions showed a decline from pH 5.5 to pH 9.0.

Animals↗
PubMed1995

Purification and characterization of an Ins(1,3,4,5)P4 binding protein from pig platelets: possible identification of a novel non-neuronal Ins(1,3,4,5)P4 receptor.

A novel Ins(1,3,4,5)P4-binding protein has been purified to apparent homogeneity from solubilized membranes derived from pig platelets. It has a high affinity for Ins(1,3,4,5)P4 (Kd 6.3 +/- 0.4 nM), a Bmax of 2.5-6.0 nmol/mg of protein, and a high specificity for Ins(1,3,4,5)P4 [Kd values for Ins(1,3,4,5,6)P5, InsP6, GroPtdIns(3,4,5)P3, Ins(1,4,5)P3, Ins(3,4,5,6)P4 and L-Ins(1,3,4,5)P4 of 85.0 +/- 4.1 nM, 800.0 +/- 20.2 nM, 65.6 +/- 2.6 nM, > 10 microM, 793.3 +/- 55.6 nM and 81.0 +/- 5.9 nM respectively]. The protein has an apparent molecular mass of 104 kDa, suggesting that this peripheral tissue protein may be different from Ins(1,3,4,5)P4 binding proteins previously isolated from neuronal tissues.

Animals↗